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What is NMR?
Nuclear Magnetic Resonance
Innovation with Integrity
NMR
Nuclear Magnetic Resonance
You may have heard the term NMR – nuclear magnetic resonance – but how
much do you actually know about it? NMR is a valuable analytical technique
for the laboratory scientist, while a similar, although not absolutely identical,
technique, magnetic resonance imaging (MRI), has become an indispensable
medical diagnostics tool.
In modern hospitals, magnetic resonance imaging is
a familiar diagnostics tool. Anybody who has undergone an MRI scan will already have seen the amazingly
detailed images it can generate of the inside of the
body, helping doctors to make an informed diagnosis
without the need for invasive – and costly – exploratory
surgical procedures, for example arthroscopic examination of the knee. NMR, on the other hand, is probably
still something of a mystery to most people outside of
the scientific laboratory.
NMR was first described by Isidor Rabi as far back as
1938, and has grown to become a well-established,
powerful physical tool for the investigation of matter
through the measurement of nuclear magnetic interactions. The benefits of the technique as a probe to
study ordinary matter really began to be appreciated in
1946, when Harvard University’s Edward Mills Purcell
detected the first solid-state NMR signal in 1 kg of paraffin wax. At almost exactly the same time, Felix Bloch
at Stanford University successfully performed the first
liquid-state NMR experiment in water; both achievements were later recognized with the joint award of the
1952 Nobel Prize in Physics. Incredibly, no fewer than
eight Nobel Laureates from around the globe have been
honored in physics, chemistry and medicine for the discovery, development and application of NMR!
Today, a plethora of NMR experimental methods provide access to a wealth of in-depth information about
the structure and dynamics of unbelievably complex
molecules, and the versatility of the technique has
allowed it to be employed in countless applications
across diverse scientific fields, among them physics,
chemistry, biology, biochemistry, materials science,
food, geology, pharmaceutical research and medicine.
With NMR methods and associated technologies continuing to be developed in universities and industry, the
future possibilities are endless.
Why do we need NMR?
Molecules are essentially arrangements of atoms, with
varying structures depending on which atoms – and
how many – are present and the way in which they are
bonded to each other. But why do we want to know
about molecular structure?
The structure of a molecule strongly influences a substance’s physical and chemical properties and, as a
result, how it reacts with other molecules or affects
living organisms. For example, ethanol – also known as
absolute alcohol – and dimethyl ether are composed of
the same type and number of atoms; two carbon, six
hydrogen and one oxygen. However, the structures,
and therefore the properties, are quite different. Ethanol is a liquid, while dimethyl ether is a poisonous gas!
Ethanol and dimethyl ether clearly demonstrate one
reason that chemical structure is so important, but this
knowledge is also crucial in the field of organic synthesis to successfully create a synthetic equivalent of an
existing substance, or to synthesize novel compounds.
An in-depth understanding of molecular structure is
particularly important in the chemical and pharmaceutical industries for the production of materials such as
nylon and plastic, and for drug discovery. For scientists,
NMR is a key tool that provides this vital information.
NMR can also be used to determine the structure of
proteins – very large, biologically important molecules
that regulate virtually all of the body’s functions and
the biochemical processes that are essential to life.
For example, the protein insulin regulates blood sugar
levels; another protein, hemoglobin, carries oxygen in
the blood around the body; and antibodies – or immunoglobulins – enable the immune system to identify
and neutralize foreign compounds such as bacteria and
viruses. Such molecules and the processes associated
with them must be studied in detail and a thorough
understanding acquired if new medicines or therapies
are to be developed. This requires knowledge of not
only the molecular structure, but also the dynamics of
proteins. NMR is the only analytical method with the
capability to provide both structural and dynamic information of biological molecules in their physiological
environment with atomic resolution.
H3C
O
CH3
Dimethyl ether
Ethanol
Atoms, nuclei and molecules
To understand NMR, we need to first consider the
relationship between atoms, nuclei and molecules.
To date, 118 elements have been identified, including
hydrogen, carbon and oxygen. Elements are pure
chemical substances consisting of a single type of
atom distinguished by its atomic number. Although
the Ancient Greeks assumed atoms – named after the
Greek word for indivisible, atomos – are the smallest
units of matter, they can actually be subdivided into even
smaller particles. Each atom consists of a positively
charged nucleus containing protons (positively charged)
and neutrons (uncharged), surrounded by a cloud of
negatively charged electrons.
Molecules – collections of atoms bonded together in
a unique molecular structure – exist in a wide range
of shapes and sizes, from small compounds such as
water through to much larger compounds consisting
of hundreds of thousands of atoms. In each case,
the molecular structure determines the physical and
chemical properties of the compound. To gain an
idea of scale, consider a molecule of water, a simple
compound consisting of two hydrogen atoms bonded to
one oxygen atom. A single raindrop contains 4 x 101021
molecules of water. If a water molecule were the size
of a football, then a raindrop would be a similar size to
the earth. It’s a bit like looking for the proverbial needle
in a haystack. Molecules, even very large ones, are so
minute that they cannot be seen even with the most
powerful conventional microscopes; other methods are
required to establish molecular structure.
NMR is one of the best-suited methods for determining
molecular structure. When a molecule is exposed to an
external magnetic field, each atom will feel the effect of
a marginally different, modified field, depending on the
magnetic shielding effects of the neighboring electric
charges – the nuclei and electrons.
This means that the magnetic field experienced by each
atom is a local property of the molecule, and is dependent
on its geometry. NMR has the capability to measure the
response of an atom to the local magnetic field – modern
instruments are sensitive to local magnetic field variations
as small as one part in a billion – from which information
can be determined about the molecular structure; even
the structures of very large, complex molecules, such
as proteins, can be determined with incredible accuracy.
This is particularly important in the field of drug research,
where contributions from Professor Kurt Wüthrich from
the ETH Zürich were recognized by the award of the
2002 Nobel Prize for Chemistry.
How does NMR work?
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NMR takes advantage of the influence of a strong magnetic field. Many atomic nuclei possess a physical property known as spin. Put simply, this is the rotation of a
nucleus about its own axis. Rotating, electrically charged
nuclei generate a magnetic field and therefore carry a
magnetic moment – a measure of an object’s tendency
to align with a magnetic field – represented graphically
by the green and red arrows. This can be
thought of as a tiny bar magnet rotating
around a magnetic axis like a spinning top.
Similarly to compass needles, the nuclei
can be manipulated by means of magnetic
fields. Typically, macroscopic magnetization
does not occur with an ensemble of atoms, as each
of these small ‘magnets’ is randomly oriented, pointing in completely different directions. This means that
the magnetic fields generated cancel each other out.
However, nuclear magnetic moments can be aligned
by exposure to a sufficiently strong magnetic field. The
resulting macroscopic ensemble magnetization is what
makes NMR possible.
An NMR experiment involves placing a sample of the
substance to be examined in a strong static magnetic
field. This forces the nuclear magnetic moments to align
parallel to the applied field – represented by the yellow
arrows – acquiring macroscopic magnetizaSouth
tion. The sample is then said to polarized,
or magnetized. While the magnetic fields in
modern NMR spectrometers can be as large
as 24 Tesla, the total fraction of aligned spins
North
is of the order of just one in 10,000, and these
few nuclei must provide sufficient information about the
molecules under investigation. However, as the nuclear
magnetic moments are very weak, they cause virtually
no interference with the molecular degrees of freedom.
The significance of this is that the molecular structure is
not altered by NMR spectroscopy.
It is a remarkable physics fact that a broad range of different resonance frequencies can all be carried by a single
radio wave pulse, which can be as short as a
South
microsecond; this is the concept behind Fourier transform NMR (FT-NMR). The sample
is irradiated with a short electromagnetic
pulse, characterized by a broad spectrum
North
of well-defined radio waves, or resonance
frequencies. This has the effect of forcing the nuclear
magnetic moments from their equilibrium position parallel to the magnetic field and into a perpendicular plane.
Each of the various nuclei in a molecule will respond to a
specific resonance frequency, and this provides information about the type of atoms present in the molecule and
their positions.
The deflected nuclear magnetic moments start a rotational motion, called a precession, around the static magnetic field (yellow arrows). The frequency of
this precession, ω, depends mainly on the
strength of the magnetic field and the type
of atom and, to a very small but detectable
degree, the atom’s position in the molecule.
The characteristic precession frequencies
of the nuclei in a molecule contain valuable information
that can be used to deduce the structure of the molecule itself. A means of measuring these frequencies is
required: the NMR spectrometer.
In an NMR spectrometer, the sample is placed in a small
coil of wire. The rotating (precessing) magnetic moments
induce an alternating voltage in the coil which can be likened to a bicycle dynamo; the NMR signal. This NMR
signal is often the overlay of many oscillating electric
signals, with the frequency of each oscillating signal corresponding to the precession frequency of the magnetic
moment that generates it. As the magnetic moments
spontaneously and progressively return to
their equilibrium state parallel to the vertical
axis, the amplitude of the observed voltage
decays. The tiny initial sample magnetization means that the NMR signal is extremely
weak; by analogy, if the power of the input
radio frequency used to stimulate the nuclei was the
size of Mount Everest, the output signal measured in an
NMR experiment would be 10,000 times smaller than
the diameter of a human hair. Extremely sophisticated
electronics are required to detect the signal.
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Finally, Fourier transform – a mathematical manipulation
which can unravel all the individual frequencies – allows
the NMR signal to be displayed as an NMR spectrum,
where the characteristic frequencies of the
precessing nuclear magnetic moments are
shown as distinct peaks known as resonance lines. For example, the NMR spectrum of ethanol shows two distinct families
of peaks. Their positions, or frequencies, are
determined by the atomic structure of the molecule,
enabling the molecular structure of ethanol to be determined.
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The NMR spectrum: Chemical shift
and spin-spin coupling
Shift
In addition to the field of the magnet, the exact resonance
frequency of individual atoms is affected by the chemical
environment. These frequency differences are generally
in the range of parts per million of the basic resonance
frequency of any given nucleus. These differences are
also depending on the strength of the magnetic field. To
allow comparison of spectra from different instruments
a frequency independent scale the Chemical Shift has
been defined. It is expressed in parts per million (ppm),
according to the equation:
solvent
δ (ppm)= (frequency of signal (Hz) - frequency of reference (Hz)) /frequency of reference (MHz)
Compounds used as chemical shift references have been defined for all NMR active nuclei. For example the chemical shift of tetramethylsilane is assigned to zero ppm in 1H and 13C spectra.
Coupling
As well as interacting with the magnetic field, nuclei interact with
each other through the chemical bonds of the molecule, generating
the fine structure in the NMR signal. This is known as coupling, and
is extremely useful for characterizing neighboring atoms in a molecule. Each spin can be affected by spins a few bonds away according to the general rule 2nI+1, where n is the number of spins and I
is the spin quantum number. For spin = ½ nuclei such as protons
this is simplified to n+1. One neighboring spin leads to a splitting of
the resonance into two lines, two spins result in three lines etc. The
intensity of the lines follows the coefficients of Pascal’s triangle.
Quantitation
NMR is a primary ratio method of
analysis. The area of each signal is
directly proportional to the number
of atoms responsible for said signal.
Signal areas are determined by integration or deconvolution of signals.
Quantiation of different components
in a mixture can also be obtained
with internal or external references
allowing the direct determination of
concentrations.
NMR instrumentation
Early NMR experiments were carried out using a technique known as continuous wave (CW). A constant
magnetic field was maintained, and the radio signal
swept through the appropriate frequency range. This
sequentially brought each nucleus into resonance. As
the experiment progressed, the spectrum was drawn
on a chart recorder in real time. This meant that resolution could only be improved by slowing down the frequency sweep, and the corresponding pen movement.
In the 1970s, continuous wave was replaced by Fourier
transform NMR, described above. FT­
NMR – discovered by Professor Richard Ernst from the ETH Zürich,
who received the Nobel Prize for Chemistry in 1991 for
his efforts – resulted in a considerably faster and more
sensitive technique on which hundreds of NMR experiments have since been based.
FT-NMR offers the advantages of:
„„ Signal
averaging; repeated measurements to
improve signal-to-noise ratios
„„ Resolution; the free induction decay (FID) – a composite of all the NMR signals – can be sampled with
a large number of points to ensure the fine structure
is resolved
„„ Versatility; FT spectroscopy is not limited to single
pulse experiments, enabling manipulation of the
various spins in a given molecule.
The most essential component of an NMR spectrometer is the magnet, which generates the field in which
a sample’s nuclear magnetic moments are aligned and
magnetization created. Only a very small fraction of the
nuclear spins in a sample can be effectively aligned to
the magnetic field, which limits the degree of magnetization that can be achieved; as magnetization is approximately proportional to the intensity of the magnetic
field, it is crucial to apply the strongest possible field.
field of the Earth. These magnets are immersed in a
cryostat-controlled liquid helium bath, operating at temperatures in the region of 2 to 4 K, which is about -270
°C. A system of coils built into the spectrometer’s probe
acts as a physical interface between the sample and the
magnet, and generates radio wave pulses. The sample,
either liquid or solid, is placed into the probe, where it
is at the center of the radio wave coils and the electronics required for signal detection. The electronics of
modern probes – known as cryoprobes – allow cooling
to between 10 and 20 K using an external cooling unit.
This reduces electronic noise, helping to increase sensitivity and enable the detection of small signals that
would otherwise be invisible. The spectrometer also
contains a pre-amplifier to boost the initial signal, as
well as a control console that houses the sophisticated
electronics required to generate the radio wave pulses
and detect the NMR signal. Data acquisition and processing are software controlled.
Developing new experimental opportunities
FT-NMR has opened the door to exciting new experimental opportunities. FT-NMR is considerably faster
and more sensitive than its predecessor, continuous
wave, and hundreds of NMR experiments have been
developed based on this technique. One particularly
powerful development is two dimensional (2D) NMR,
which allows the structure of even large molecules,
with perhaps thousands of atoms, to be resolved, typically with acquisition times between a few minutes
and a few hours, depending on the instrumentation
and quality required. One example is 2D NMR of a steroid hormone, a complex molecule with many strongly
overlapping resonance lines, where the technique has
proved very effective.
Today’s NMR spectrometers use superconducting magnets capable of producing magnetic fields of up to 24
Tesla – almost a million times greater than the magnetic
2D NMR spectrum of approximately 200 micrograms of a steroid hormone
Summary
Bruker BioSpin
[email protected]
www.bruker.com
© Bruker BioSpin 01/15 T153283
This article outlines the theory behind NMR spectroscopy, providing background knowledge for scientists
wishing to learn about the technique. The powerful
NMR technology has been applied to many different
applications, among them chemical analysis, structural
biology, drug development and materials science, as
well as medicine, and has proved invaluable in the laboratory, while its clinical counterpart, MRI, has become
indispensable in the hospital environment. Interest in
NMR has never been greater.